Hemolytic Anemia: Autoimmune Hemolytic Anemia and

PNH

Marc Zumberg MD, FACP Associate Professor of Medicine

University of Florida May 2010

Hemolytic Anemia--Causes

Hereditary RBC Membrane

HS, HE, pyropoikilocytosis RBC Metabolic Defects

EM pathway HMP shunt Nucleotide synthesis

Hemoglobin Defects Thalassemia Abnormal variants

Acquired Immune

Autoimmune Isoimmune Drug

RBC fragmentation syndromes PNH Secondary

Renal and liver disease Misc

Drugs, infections, chemicals, toxins, physical agents

Laboratory approach to the patient with suspected hemolytic

anemia

Laboratory CBC Reticulocyte Count Blood smear LDH Bilirubin-direct and indirect Haptoglobin Urinalysis

Laboratory evaluation of hemolytic anemia

Direct Coombs

Positive Negative

Coombs + Warm autoimmune hemolytic anemia

Cold agglutinin disease

Drug induced

Paroxysmal Cold Hemoglobinuria

Coombs -

Hemoglobinopathies Enzymopathies Membrane Defects Microangiopathic Drugs Toxins/Wilsons disease PNH Etc.

Autoimmune Hemolytic Anemia (AIHA)-Classification

Warm-reacting antibodies (optimally bind red blood cells at

37C Idiopathic Secondary

Autoimmune disorders Immunodeficiencies Lymphoproliferative disorders Nonlymphoid malignancies Viral infections

Mixed warm and cold antibodies

Drug Induced

Cold-reacting antibodies (optimally bind red blood cells

Audience Response Question: Coombs test

Which of the following statements about the Coombs test and the blood bank evaluation of autoimmune hemolytic anemia is most accurate?

A.)A positive Coombs test implies at least low grade hemolysis

B.)Cold agglutinins are typically IgG positive on the Coombs test

C.)The thermal amplitude of a cold agglutinin is the best predictor of clinical significance

D.)Autoadsorption of autoantibodies are useful to define the specificity of the antibody

E.)The eluate of concentrated IgG from patients with warm autoimmune hemolytic anemia is used to rule out alloantibodies

Direct Coombs test Detects antibody coating the RBC surface

Positive test isnt necessarily diagnostic of hemolysis As many as 0.1% of healthy blood donors are positive 1-2% of hospitalized patients are positive

Degree of hemolysis doesnt always correlate with degree of positivity

Reardon. Am J Clin Pathol 2006:125(supl1) S71-S77

Direct Coombs Test

IgG

C3

30-40% IgG only 10% C3 only 40-50% mixed

37 C

Patterns IgG C3

Warm AIHA

(67%) ++ +

(20%) + -

(13%) - +

Cold agglutinin disease

- +

Paroxsymal Cold Hemoglobinuria

- +

Adapted from Petz and Swisher. Immunology and Its RelaEon to Blood Transfusion. In:Clinical PracEce of Transfusion Medicine 3rd ed NY, NY. Churchill-Livingstone. 1996. P.49

Elution: Detection of specificity of the autoantibody

Elute off IgG from patient RBCs Incubate with reagent RBCs to test for activity and

specificity of the antibody

Antibody most commonly reacts to a full RBC panel with similar agglutination strengths

Less commonly may show a relative specificity within the Rh system such as the e antigen (WAHIA) or I (cold agglutinin)

Difficulty performing a Type and Screen

+ ++ +

Autoadsorption: Detection of Alloantibodies

Goal is remove the autoantibody from the serum to allow the detection of alloantibodies 32% of patients with AIHA have alloantibodies 1 ml of packed autologous RBCs treated to remove the Ab

and than incubate with patients serum at 37C

Reardon AM J Clin Pathol 2006;125(Suppl 1):S71-77

Transfusion in AIHA Proceed with caution-consider risk/benefit

ratio Transfused blood often has a short half-life Use phenotype matched blood if available

Rh groups, Kell, Duffy, Kidd antigens If antibody shows specificity for a given antigen use

antigen negative blood

If cold agglutinin disease transfuse through a blood warmer

Reardon AM J Clin Pathol 2006;125(Suppl 1):S71-77

Case 1 A 63 year-old previously healthy man is admitted with angina and dyspnea on exertion. He has had a lack of energy for 2 months and has also complained of some intermittent yellowing of his eyes. No prior blood counts are available. He is taking no medications and does not recall any recent illness. He denies alcohol consumption, tobacco, or illicit drug use. Laboratory data and blood smear are as follows: Hct 26% MCV 103 fL WBC count 5900/ul Retic count 7.1 % LDH 949 IU/L Coombs IgG+++, C3+

Warm Autoimmune Hemolytic Anemia (W-AIHA)

IgG panagglutinating antibody directed against public epitope often on the Rh system

Optimally bind red cells at 37C

Primarily removed by Fc receptor macrophages in the reticuloendothelial system

Partial phagocytosis leads to spherocytes removed by the spleen

Less commonly or weakly fixes complement

Packman CH. Blood Rev. 2008 Jan;22(1):17-31. PMID: 17904259

spherocyte

nucleated RBC

Positive Coombs Test

Diagnosis: Warm Autoimmune hemolytic Anemia

Investigation Rule out lymphoproliferative disorder

Consider bone marrow aspirate and biopsy

Rule out autoimmune disorder ANA, etc

Rule out immunodeficiency Quantitative immunoglobulins

Rule out drugs

Warm autoimmune hemolytic anemia-Treatment

Treat underlying disease if identified

Steroids first line (1mg/kg) for one to three weeks Interfere with ability of macrophages to clear IgG coated

RBCs Decreases antibody production 60-85% initial response (20% CR), but frequent relapses Initial quick taper, than slowly when down to 20mg (for

2-3 months) Pulses of high dose glucocorticoids may be useful in some

who fail King. Semin Hematol. 2005; 42:131-136

Kessler. Clin Advances in Hemaotol Onc. 2008; 6(10):739-41

Warm autoimmune hemolytic anemia-Treatment

Splenectomy

Consider in two-three weeks if no response to steroids 2/3 respond, but relapses occur Ensure immunizations and education about infectious risk

Pneumoccus, meningococcus, and hemophilis influenza type B ??? Prophylactic antibiotics

Higher incidence of post-splenectomy venous thrombembolism and pulmonary hypertension

High incidence of antiphospholipid antibodies

Kessler. Clin Advances in Hemaotol Onc. 2008; 6(10):739-41 Crary. Blood. 2009 Oct 1;114(14):2861-8.

Rituximab Chimeric monoclonal antibody targeting

CD-20 on mature B Cells

Bussone . AM J Hematol. 2009: 84:153-157

Median time to response 6 weeks (2-16)

Treatment-Other 10% refractory to both steroids and splenectomy Other immunosuppressants

Immuran Cyclophosphamide Cyclosporine Danazol IVIG

Not as effective as in ITP

Paucity of randomized trials to suggest which agent is superior

Responses may be delayed several months Kessler. Clin Advances in Hemaotol Onc. 2008; 6(10):739-41

Case 2 A 63 year-old previously healthy male complains of fatigue and lack of energy for 2 months. He denies a history of fever, chills, night sweats or weight loss, but reports painful blue digits in the cold. A routine CBC drawn 1 year ago was normal. He is taking no new medications and does not recall any recent illness. He denies alcohol consumption, tobacco, or illicit drug use Laboratory data and blood smear are as follows: Hct 26% MCV 133 fL WBC count 5900/ul Retic count 7.1 % LDH 949 IU/L Coombs IgG-, C3++

Cold agglutinin Disease

IgM antibody optimally binds to RBCs at lower temperatures Fixes complement

Direct lysis of red blood cells Removal of C3b-coated RBCs by the liver

Titers

Blood Bank evaluation-Thermal Amplitude Test

Keep blood at 37C from point of bedside collection to testing initial screening is often at 20C or room

temperature

Reardon. Am J Clin Pathol 2006:125(supl1) S71-S77

Cold Agglutinin Disease: Diagnostic Criteria

1.) Clinical evidence of an acquired hemolytic anemia

2.) Positive Coombs test using anti-C3

3.) Negative Coombs test using anti-IgG

4.) Presence of cold agglutinin with reactivity up to 30C

5.) Cold agglutinin titer at 4C > 256

Petz. Blood Reviews. 2008; 22: 1-15

Spurious marked elevation of MCV, MCHC may occur Agglutination will abate with warming

RBC agglutination

Cooling of blood during passage through acral parts of the body allows cold agglutinins to bind to the RBC leading to agglutination,

complement binding, and hemolysis

Berntsten. Hematology. 2007; 12(5): 361-70

Investigation of Cold Agglutinin Disease

Consider infectious etiologies such as Mycoplasma or Epstein-Barr virus

Consider lymphoma or other lymphoproliferative disorders Serum protein electrophoresis (SPEP) Bone marrow biopsy if no obvious infection Consider radiologic imaging

Berntsten. Hematology. 2007; 12(5): 361-70

Cold Agglutinin Disease Monoclonal band can be detected in the majority of

patients on serum protein electrophoresis/im